Lignin impairs Cel7A degradation of in vitro lignified cellulose by impeding enzyme movement and not by acting as a sink

Background Cellulose degradation by cellulases has been studied for decades due to the potential of using lignocellulosic biomass as a sustainable source of bioethanol. In plant cell walls, cellulose is bonded together and strengthened by the polyphenolic polymer, lignin. Because lignin is tightly linked to cellulose and is not digestible by cellulases, is thought to play a dominant role in limiting the efficient enzymatic degradation of plant biomass. Removal of lignin via pretreatments currently limits the cost-efficient production of ethanol from cellulose, motivating the need for a better understanding of how lignin inhibits cellulase-catalyzed degradation of lignocellulose. Work to date using bulk assays has suggested three possible inhibition mechanisms: lignin blocks access of the enzyme to cellulose, lignin impedes progress of the enzyme along cellulose, or lignin binds cellulases directly and acts as a sink. Results We used single-molecule fluorescence microscopy to investigate the nanoscale dynamics of Cel7A from Trichoderma reesei, as it binds to and moves along purified bacterial cellulose in vitro. Lignified cellulose was generated by polymerizing coniferyl alcohol onto purified bacterial cellulose, and the degree of lignin incorporation into the cellulose meshwork was analyzed by optical and electron microscopy. We found that Cel7A preferentially bound to regions of cellulose where lignin was absent, and that in regions of high lignin density, Cel7A binding was inhibited. With increasing degrees of lignification, there was a decrease in the fraction of Cel7A that moved along cellulose rather than statically binding. Furthermore, with increasing lignification, the velocity of processive Cel7A movement decreased, as did the distance that individual Cel7A molecules moved during processive runs. Conclusions In an in vitro system that mimics lignified cellulose in plant cell walls, lignin did not act as a sink to sequester Cel7A and prevent it from interacting with cellulose. Instead, lignin both blocked access of Cel7A to cellulose and impeded the processive movement of Cel7A along cellulose. This work implies that strategies for improving biofuel production efficiency should target weakening interactions between lignin and cellulose surface, and further suggest that nonspecific adsorption of Cel7A to lignin is likely not a dominant mechanism of inhibition. Supplementary Information The online version contains supplementary material available at 10.1186/s13068-023-02456-3.

Binding locations of Qdot-labeled Cel7A were recorded for 50 seconds before being washed out and Basic Fuchsin was added to determine locations of lignin deposition.A Pearson's correlation coefficient of 0.037 was calculated, as described in Methods.This value indicates no correlation between Cel7A binding and lignin, which differs from previous results with BSA added to the flow cell as shown in Figure 3.This shows the BSA washes may affect the Cel7A binding to lignin, but the lignin still does not appear to act as a sink where a larger positive correlation would be expected.

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Figure S1 (Related to Figure 2): All CA concentrations used in data set for lignin polymerized onto acetobacter cellulose in vitro.First column: cellulose only; second column: lignin only.Third through fifth columns: cellulose with lignin polymerized from different concentrations of CA.Top row are interference reflection micrographs, scale bar = 10 µm.Bottom row are scanning electron micrographs, scale bar = 100 nm.

Figure S2 (
Figure S2 (Related to Figure 2): Lignin generated in vitro deposits heterogeneously onto acetobacter cellulose.Scanning electron micrograph of 3 mM CA sample, with lignin false-colored yellow.Areas in the middle of the image display highly lignified regions of cellulose with sheets of lignin (shown in yellow) covering significant areas of the cellulose surface In contrast, areas in the upper left and bottom right of the image contain less lignin on the cellulose surface, with some regions appearing to have nearly no visible lignin and appearing similar to the cellulose-only samples.

Figure S3 :
Figure S3: Pure in vitro polymerized lignin has similar Cel7A binding properties to lignin polymerized onto cellulose.Lignin was polymerized in the absence of cellulose, following the same procedures as the lignocellulose samples.A drop of lignin solution and a drop of purified cellulose solution were then placed together onto a glass slide.A flow cell was constructed and placed inverted in an oven at 65°C for 30 minutes to dry the lignin and cellulose

Figure S4 (
Figure S4 (Related to Figure 3): BSA reduces the binding of Cel7A to lignin.9 mM CA lignocellulose was adsorbed to the slide without the addition of BSA to the flow cell to determine if BSA affects Cel7A binding to lignin.

Figure S5 (
Figure S5 (Related to Figure 5): Line scans across the lignocellulose surfaces show an increase in Basic Fuchsin fluorescent signal in the TIRF channels on the lignified cellulose samples compared to cellulose-only samples.Yellow lines in the IRM and TIRF images show the location of the line scan for each sample.The fluorescence intensity across the cellulose surface for the cellulose-only sample is similar to the intensity on the glass surface, indicating no lignin is present in the sample.The 0.11 mM and 0.33 mM CA samples display an increase in fluorescence intensity across the lignocellulose surface compared to the glass surface, signifying the presence of a thin film of lignin on the cellulose surface.The line scans for the lignified samples also show periodic spikes, corresponding to lignin aggregates that can be seen by eye in the fluorescence image.